The appendicular skeletal system represents a paradigm for development of skeletal elements with unique shapes and locations. Moreover, this system provides a platform to study how bone/cartilage in the same position develops into distinct shapes in two types of limbs, namely the forelimbs and hindlimbs. Elucidating how these skeletal elements are generated from progenitor cells is crucial for understanding the development of the appendicular skeletal system. During embryonic development, the posterior growth zone, the structure located at the posterior of the body, grows and posteriorly extends the body. This process, called the body extension, progressively generates progenitor cells for various tissues, including the lateral plate mesoderm (LPM). Cells in the LPM give rise to the limb bud mesenchyme, which expand and undergo region-specific growth and differentiation, leading to progressive morphogenesis of skeletal elements from the most proximal (stylopod) to the middle (zeugopod) and distal (autopod) region. We obtained preliminary data supporting the idea that genetic systems earlier than limb bud formation regulate generation and expansion of progenitor cells for subsequent development of the appendicular skeletons. Conditional inactivation of Sall4, which encodes a zinc finger containing protein, prior to limb outgrowth, resulted in the loss of proximal-anterior skeletal elements only in the hindlimb, such as the femur, tibia and anterior digits. We also obtained preliminary data, suggesting that cells expressing Isl1, encoding a LIM homeodomain protein, in the hindlimb-forming region contribute to the formation of posterior-distal skeletal elements in hindlimbs, such as fibula and posterior digits. These results suggest that genetic systems by Isl1 and Sall4 operate in the LPM and regulate hindlimb progenitor cells to develop into specific skeletal elements. Furthermore, our preliminary data suggest that, in an earlier, upstream event, combined function of Sall4 and its homolog, Sall1, regulates body extension to generate hindlimb progenitors. The goal of the proposal is to determine the genetic and molecular mechanisms of Sall4, Isl1 and Sall1 operating in progenitor cells. We will have three specific aims for this goal. Aim 1 will test the hypothesis that Sall4 regulates proliferation and survival of hindlimb progenitors through regulation of the cyclin-dependent kinase inhibitor genes. Aim 2 will test the hypothesis that distinct regulation of ss-catenin by Sall4 and Isl1 controls proliferation and/or survival of hindlmb progenitor cells. Aim 3 will test the hypothesis that the combined function of Sall1 and Sall4 controls the formation of the hindlimb progenitors in the LPM through regulation of body extension. Completion of this project will reveal a novel concept that genetic systems regulate the expansion of progenitors in the LPM for development of the appendicular skeletal system in later stages. Elucidating this novel mechanism that regulates appendicular skeletal development will significantly advance our current understanding of appendicular skeletogenesis